| Four fragments -the cDNA and DNA fragments of Lectin gene,the cDNA and DNA fragments of Pectate Lyase gene were obtained from the fruit of Musa Acuminata AAA SPP. by the method of RT-PCR and PCR .They were ligated with pGEM-T Easy Vector respectively which was used to transform E.coli DH5 a later. After digestion and PCR assay,Recons were obtained and sequenced .The result of sequence analysis indicated that the length of cDNA and DNA fragments of Lectin gene and the length of cDNA and DNA fragments of Pectate Lyase gene were 508 bp,575 bp,1,316 bp and 1,858 bp respectively.The DNA fragment of Lectin gene had a intron,whose length was 67 bp .The DNA fragment of Pectate Lyase gene had 3 introns,whose lengths were 90 bp,83 bp,and 370 bp respectively. All these four introns' sequences followed the rule of GT-AG.The nucleotide acid sequences of the four fragments had more than 95% homology with Lectin gene (AF001527) and Pectate Lyase gene (x92943) which were submitted to GenBank . At the same time,the Amino Acid sequences that were induced from the cDNA and DNA fragments of Lectin gene had more than 93% homology with the Amino Acid sequence of Lectin gene (AF001527 ).The expression of banana Lectin gene and Pectate Lyase gene in different tissues and on different ripening stages of fruit was studied. The Total RNA of 12 tissues was extracted from banana's root,leaf,fruit peel and pulp on different ripening stages of post-harvest fruit. The results indicated that the Pectate Lyase gene was specificly expressed in four tissues which were the fruit peel of ripening stage 4 and 5,the pulp of ripening stage 3 and 4 respectively. It also showed that Lectin gene was specificly expressed in seven tissues which were the fruit peel of ripening stage 2,3,4 and the fruit pulp of ripening stage 2,3,4,5. This research demonstrated that both the two genes had tissue specificity and were specificly expressed on some ripening stages. Furthermore,it confirmed that the ripening stages of expression in fruit peel and pulp were also different.After qualitative study,there was a quantitative study on these two genes' expression. The procedure as followed:First,the expression of pectate Lyase gene was studied through quantitative PCR method. The result indicated that there was a maximum of the gene'sexpression in the pulp of stage 4,and then the pulp of stage 3,the fruit peel of stage 5,the fruit peel of stage 4 sequentially.Secondly,the expression of Lectin gene was relatively studied by the method of competitive PCR. The result showed that the fruit peel of stage 2,3,4,the pulp of stage 2,3,4,5 had 0.012,665,0.019,149,0.001,564,2.739,199,2.963,732,4.879,877 and 3.584,009 ng transcript/ u g Total RNA respectityely.According to the results of correlected studies,the applicated PCR method,especially the competitive PCR method which was used to verify the expression of these two genes was discussed.Finally,The DNA fragment of Lectin gene was submitted to GenBank,and the GenBank Accession Number was:AY103481... |
